The ryanodine receptor (RyR) is a prototypical ion channel that mediates Ca2+ release from the endoplasmic/sarcoplasmic reticulum, and thereby plays important roles in intracellular Ca2+ signalling in eukaryotic cells. Instances of local Ca2+ release from clusters of RyRs, in the form of "Ca2+ sparks", constitute the elementary Ca2+ signalling events in many types of cells. Ironically, the nature of Ca2+ sparks remain elusive, as known properties of RyRs in vitro cannot fully account for the genesis of Ca2+ sparks. Using the "Ca2+ sparklets"ref produced by single L-type channel Ca2+ influx as the calibration standard, here we show that Ca2+ release current associated with a spark can be split into subtler quantal units, each from the all-or-none opening of a single RyR. The quantal analysis further demonstrated that interaction of RyRs in multi-quantum sparks confers an unusual, thermodynamically irreversible behavior to RyR gating in vivo. Furthermore, spark rise time is inversely related to the number of quanta involved, indicating the presence of a strong negative feedback component in the coupling of RyRs. These findings afford a novel view of nanoscopic interactions of Ca2+ release channels in intact cells.